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1.  High-throughput plasmid DNA purification for 3 cents per sample. 
Nucleic Acids Research  1999;27(24):e37.
To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high throughput, low degree of technical difficulty and extremely low cost have made it the plasmid DNA preparation method of choice in both our expressed sequence tag (EST) and genome sequencing projects. Here we report the details of the method and describe its application in the generation of more than 700 000 ESTs at a rate exceeding 16 000 per week.
PMCID: PMC148760  PMID: 10572189
2.  Representation of cloned genomic sequences in two sequencing vectors: correlation of DNA sequence and subclone distribution. 
Nucleic Acids Research  1997;25(15):2960-2966.
Representation of subcloned Caenorhabditis elegans and human DNA sequences in both M13 and pUC sequencing vectors was determined in the context of large scale genomic sequencing. In many cases, regions of subclone under-representation correlated with the occurrence of repeat sequences, and in some cases the under-representation was orientation specific. Factors which affected subclone representation included the nature and complexity of the repeat sequence, as well as the length of the repeat region. In some but not all cases, notable differences between the M13 and pUC subclone distributions existed. However, in all regions lacking one type of subclone (either M13 or pUC), an alternate subclone was identified in at least one orientation. This suggests that complementary use of M13 and pUC subclones would provide the most comprehensive subclone coverage of a given genomic sequence.
PMCID: PMC146865  PMID: 9224593
3.  Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans. 
Molecular and Cellular Biology  1988;8(9):3627-3635.
Caenorhabditis elegans has 12 tRNA(UGGTrp) genes as defined by Southern analysis. In order to evaluate the function of the individual members of this multigene family, we sought to recover amber (UAG)-suppressing mutations from reversion experiments with animals carrying amber mutations in a nervous system-affecting gene (unc-13) or a sex-determining gene (tra-3). Revertants were analyzed by Southern blot, exploiting the fact that the CCA to CTA change at the anticodon creates a new XbaI site. Five different members of the tRNATrp gene family were identified as suppressors: sup-7 X, sup-5 III, sup-24 IV, sup-28 X, and sup-29 IV. All five suppressor genes were sequenced and found to encode identical tRNA(UAGTrp) molecules with a single base change (CCA to CTA) at the anticodon compared with their wild-type counterparts. The flanking sequences had only limited homology. The relative expression of these five genes was determined by measuring the efficiencies of suppressers against amber mutations in genes affecting the nervous system, hypodermis, muscle, and sex determination. The results of these cross-suppression tests showed that the five members of the tRNA(Trp) gene family were differentially regulated in a tissue- or development stage-specific manner.
PMCID: PMC365418  PMID: 3221861
5.  The construction and analysis of M13 libraries prepared from YAC DNA. 
Nucleic Acids Research  1995;23(4):670-674.
Yeast artificial chromosomes (YACs) provide a powerful way to isolate and map large regions of genomic DNA and their use in genome analysis is now extensive. We modified a series of procedures to produce high quality shotgun libraries from small amounts of YAC DNA. Clones from several different libraries have been sequenced and analyzed for distribution, sequence integrity and degree of contamination from yeast DNA. We describe these procedures and analyses and show that sequencing at about 1-fold coverage, followed by database comparison (survey sequencing) offers a relatively quick method to determine the nature of previously uncharacterized cosmid or YAC clones.
PMCID: PMC306736  PMID: 7899089
6.  Germline excision of the transposable element Tc1 in C. elegans. 
Nucleic Acids Research  1991;19(20):5669-5672.
We have examined eight germline revertants generated by the excision of Tc1 from a site within the unc-22 gene of Caenorhabditis elegans. A rich variety of rearrangements accompanied Tc1 excision at this site, including transposon 'footprints', deletions of sequences flanking the insertion site and direct nontandem duplications of flanking DNA. With only modest modification the double-strand gap repair model for transposition, recently proposed by Engles and coworkers (Cell 62: 515-525 1990), can explain even the most complex of these rearrangements. In light of this model rearrangements of the target site accompanying transposition/excision may not be the end result of imprecise excision of the element. Instead, these rearrangements may be the result of imprecise repair of the double-strand gap by the host replication and repair machinery. Sequences surrounding an insertion site influence the fidelity of gap repair by this machinery. This may lead to a number of possible resolutions of a double-strand gap as documented here for a Tc1 site in unc-22.
PMCID: PMC328973  PMID: 1658738
7.  Talin requires beta-integrin, but not vinculin, for its assembly into focal adhesion-like structures in the nematode Caenorhabditis elegans. 
Molecular Biology of the Cell  1996;7(8):1181-1193.
In cultured cells, the 230-kDa protein talin is found at discrete plasma membrane foci known as focal adhesions, sites that anchor the intracellular actin cytoskeleton to the extracellular matrix. The regulated assembly of focal adhesions influences the direction of cell migrations or the reorientation of cell shapes. Biochemical studies of talin have shown that it binds to the proteins integrin, vinculin, and actin in vitro. To understand the function of talin in vivo and to correlate its in vitro and in vivo biochemical properties, various genetic approaches have been adopted. With the intention of using genetics in the study of talin, we identified a homologue to mouse talin in a genetic model system, the nematode Caenorhabditis elegans. C. elegans talin is 39% identical and 59% similar to mouse talin. In wild-type adult C. elegans, talin colocalizes with integrin, vinculin, and alpha-actinin in the focal adhesion-like structures found in the body-wall muscle. By examining the organization of talin in two different C. elegans mutant strains that do not make either beta-integrin or vinculin, we were able to determine that talin does not require vinculin for its initial organization at the membrane, but that it depends critically on the presence of integrin for its initial assembly at membrane foci.
PMCID: PMC275971  PMID: 8856663
8.  The alpha and beta subunits of nematode actin capping protein function in yeast. 
Molecular Biology of the Cell  1993;4(9):907-917.
We cloned and analyzed two genes, cap-1 and cap-2, which encode the alpha and beta subunits of Caenorhabditis elegans capping protein (CP). The nematode CP subunits are 55% (cap-1) and 66% (cap-2) identical to the chicken CP subunits and 32% (cap-1) and 48% (cap-2) identical to the yeast CP subunits. Purified nematode CP made by expression of both subunits in yeast is functionally similar to chicken skeletal muscle CP in two different actin polymerization assays. The abnormal cell morphology and disorganized actin cytoskeleton of yeast CP null mutants are restored to wild-type by expression of the nematode CP subunits. Expression of the nematode CP alpha or beta subunit is sufficient to restore viability to yeast cap1 sac6 or cap2 sac6 double mutants, respectively. Therefore, despite evolution of the nematode actin cytoskeleton to a state far more complex than that of yeast, one important component can function in both organisms.
PMCID: PMC275721  PMID: 8257793

Results 1-8 (8)